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Vietnam Journal of Science and Technology 58 (6A) (2020) 110-122 doi:10.15625/2525-2518/58/6A/15486 APPLICATION OF ULTRASONIC-ASSISTED ENZYMATIC EXTRACTION FOR POLYSACCHARIDES FROM VIETNAMESE RED GANODERMA LUCIDUM AND EXAMINATION OF ANTIOXIDANT ACTIVITY OF THE EXTRACT Nguyen Thi Kim Ngan1, Tran Do Dat1, Dang Hoang Lam1, Phan Le Thao My1, Ngo Thi Thuy Linh1, Vuong Hoai Thanh2, Nguyen Duc Viet2, Ngo Hong Thao2, Huynh Thi Thanh Tu2, Nguyen Huynh Bach Son Long4, Hoang Minh Nam2,3, Mai Thanh Phong2, 3, Nguyen Huu Hieu1, 2, 3, * 1 VNU-HCMC Key Laboratory of Chemical Engineering and Petroleum Processing (Key CEPP Lab), Viet Nam, 70000 2 Faculty of Chemical Engineering, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, Ward 14, District 10, Ho Chi Minh City, Viet Nam 3 Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Viet Nam 4 Department of Chemical Engineering, Lac Hong University, 10 Huynh Van Nghe Street, Buu Long Ward, Bien Hoa City, Dong Nai Province, Viet Nam * Email: nhhieubk@hcmut.edu.vn Received: 11 September 2020; Accepted for publication: 30 December 2020 Abstract. Commonly used in Vietnamese traditional remedies, Vietnamese red Ganoderma lucidum (G. lucidum) is an oriental fungus that has long been known for promoting health and longevity. In this study, polysaccharides (PS) were extracted from G. lucidum using ultrasoundassisted enzymatic extraction (UAEE) method, followed by the investigation of seven singlefactor experiments namely enzyme ratio between viscozyme and chitinase, total enzyme volume, pH value, extraction temperature, material-to-solvent ratio, ultrasonic power, and extraction time. Based on ultraviolet-visible spectroscopy analysis, the highest PS content could be achieved with a value of 59.71 mg/g under extraction conditions including the enzyme ratio between viscozyme and chitinase of 3:1, total enzyme volume of 100 µL, pH value of 5.5, extraction temperature of 45 material-to-solvent ratio of 1:25, ultrasonic power of 480 W, and extraction time of 30 min. The extract obtained was then evaluated for antioxidant activities by using 2,2-Diphenyl-1-picrylhydrazyl radical scavenging method, showing that the halfmaximal inhibitory concentration values were of 1727.15 µg/mL. As a result, the UAEE method could be regarded as an efficient approach for antioxidant crude polysaccharides content extraction from Vietnamese red G. lucidum. Keywords: Ganoderma lucidum, ultrasound-assisted enzymatic extraction, polysaccharides, antioxidant activities. Classification numbers: 1, 3. Application of ultrasonic-assisted enzymatic extraction for polysaccharides from Vietnamese … 1. INTRODUCTION Phytochemicals, also known as bioactive nutrient plant chemicals, have long been utilized as potentially chemo-preventive agents in food, cosmetic, and pharmaceutical industries with many health benefits [1, 2, 3]. Throughout the world, especially in temperate and subtropical locations including North and South America, Europe, and Asia, Ganoderma lucidum (G. lucidum) is a popular fungus species belonging to the family of Ganodermatacea of Polyporales and has been highly praised for its health benefits as improving longevity and possessing various biological abilities such as antioxidant, antimicrobial, antiviral, and anti-inflammatory activities [4-9]. According to previous studies, from G. lucidum, many bioactive compounds were determined encompassing triterpenoids, polysaccharides, alkaloids, fatty acids, organic acids, and polyphenols, among other substances [1, 7]. It was reported that one of the most important biologically active constituents are triterpenoids and polysaccharides (PS) of G. lucidum with more than 200 polysaccharides and 130 triterpenoids have been identified [10, 11, 12]. Extraction is defined as the first step for separation and purification process of natural compounds from natural resources [13]. For thousands of years, conventional extraction methods such as maceration, percolation, and reflux extraction methods have been used for extracting natural products with many drawbacks related to lower extraction yield, high energy cost, and toxic organic solvents [14, 15, 16]. In addition, extraction process relied strongly upon several factors including extraction methods, raw materials, and solvents [17]. Thus, the green innovations of extraction methods with the employment of ultrasound, microwave, and enzyme have ubiquitously gained scientific attention due to its efficiency, eco-friendly environment, economic, and safety [18]. Basically, ultrasound has been considered as one of the most efficient extraction methodologies because of the perturbation of plant cell walls induced by cavitation and facilitation of mass transfer as well as particle size reduction caused by mechanical and thermal effects [19]. Additionally, enzymes have been reported to improve the yield of extraction process by hydrolyzing and disintegrating plant cell walls [20]. Due to several advantages of a combination of ultrasound and enzyme for natural compound extraction such as short extraction time, high efficiency, and lower solvent quantity, ultrasound-assisted enzymatic extraction (UAEE) has been prominently considered in different extraction studies [21]. In this study, the application of UAEE method for an efficient approach for antioxidant crude PS content extraction from Vietnamese red G. lucidum was investigated, followed by the evaluation of the anti-oxidant activity of the extract obtained in comparison with ascorbic acid. 2. MATERIALS AND METHODS 2.1. Materials and chemicals Dried fruiting bodies of Vietnamese red G. lucidum in maturity stage with mature spores were stored in a closed plastic bag and were supplied by Linhchivina Co., JSC (Viet Nam). Besides, viscozyme was purchased from Novozymes, Denmark, chitinase was purchased from Sigma – Aldrich, 95.0 % and 99.5 % ethanol were purchased from Chemsol. 99.0 % D-glucose, 99.5 % sodium hydrogen phosphate (Na2HPO4), 99.5 % sodium tetraborate decahydrate (Na2B4O7.10 H2O), 99.5 % phenol, 98.0 % sulfuric acid (H2SO4), 99.0 % citric acid, and 99.7 % ascorbic acid were purchased from Xylong, China. 2.2. Sample preparation 111 Nguyen Huu Nieu, et al. The dried G. lucidum fruiting body was ground in the SEKA Z10 blender with origin from Japan. 5.0 g of Vietnamese red G. lucidum powder was dispersed in 100 mL of distilled water. Then, 100 µL of enzyme mixture of enzymes including vicozyme and chitinase with ratio value of 1:1 was added for 1 hour of incubation. The mixture was then kept under sonication condition. During the experiments, the operational parameters including the viscozyme – chitinase enzyme ratio, total enzyme volume, pH values, incubation temperature, material-tosolvent ratio, ultrasonic power, and ultrasonic time were set based on the experimental design. After filtration, the solvent was partially removed by using vacuum evaporation at 65 - 70 . The concentration was then precipitated with the addition of 100 mL 99.5 % ethanol at 4 in 12 hours. Finally, the mixture was centrifuged and the precipitate was collected and dried to obtain the crude PS. 2.3. Determination of the PS content The phenol-sulfuric acid colorimetric method was used to determine PS content with Dglucose as a standard solution. The effects of factors affecting the PS content including the viscozyme – chitinase enzyme ratio were prepared from D-glucose solution with concentration of 1000 μg/mL. Then 1 mL of each standard solution was removed and transferred to a 20 mL volumetric flask, followed by the addition of 1 mL of 5 % phenol solution and 5 mL of 98 % concentrated sulfuric acid solution. Besides, the mixture containing 1 mL of distilled water, 1 mL of 5 % phenol solution, and 5 mL of 98 % concentrated sulfuric acid was prepared for a blank solution while the extract solution was prepared by the addition of 1 mL of the extract, 1 mL of 5 % phenol solution, and 5 mL of 98 % concentrated sulfuric acid. After 30 min, the measurement of absorbance was conducted at 488 nm. The PS content was determined according to the absorbance of the extract solution and baseline. The yield of polysaccharides (mg/g) was calculated by the following equation (1): (1) where Y is the yield of polysaccharides (mg/g); C is the concentration of polysaccharides obtained from the calibrated regression equation (mg/L); V is the volume of polysaccharides solution (mL); n is the dilution factor; 10-3 is the conversion factors; m is the initial mass of Vietnamese red G. lucidum powder. 2.4. Single-factor experimental design Effects of varying extraction conditions including viscozyme – chitinase enzyme ratio (1:0, 3:1, 1:1, 1:3, and 0:1), total enzyme volume (60, 100, 140, 180, and 220 µL), pH values (4.0, 4.5, 5.0, 5.5, and 6.0), extraction temperature (40, 45, 50, 55, and 60 ℃), material-to-solvent ratio (1:10, 1:15, 1:20, 1:25, and 1:30), ultrasonic power (240, 360, 480, 600, and 720 W), and extraction time (10, 20, 30, 40, and 50 min) were determined with respect to the PS content. Throughout the single-factor experiments, one variable was changed while the other variables were kept constant. 2.5. Antioxidant activity investigation The scavenging of DPPH radicals was assayed according to a previous study [22]. 4.0 mL of sample extract was added to an equal volume of 6.0 mL of 1 mM DPPH solution with methanol. The mixture was then mixed well and allowed in the dark for 30 min at room 112 Application of ultrasonic-assisted enzymatic extraction for polysaccharides from Vietnamese … temperature before the absorbance of the mixture was read at 517 nm. The anti-oxidation activity is proportional to the disappearance of DPPH. The ascorbic acid was used as a positive control sample in DPPH anti-oxidation activity test. A different concentration of ascorbic acid (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 µg/mL) was prepared and the procedure was carried out in the same manner as for the sample extract. All the experiments were conducted in triplicate. The half-maximal inhibitory concentration (IC50) values of ascorbic acid and the extracts were calculated from the regression model of sample concentration and radical scavenging activity, which was determined by the following equation: ( ) where solution. is the absorbance of DPPH solution and (2) is the absorbance of sample 2.6. Statistical analysis The one-way analysis of variance (ANOVA) test with least significant difference (LSD) was used to statistically investigate the average yield of polysaccharides. The software package Statgraphics Centurion 18 (Statgraphics Technologies, Inc., Warrenton, VA, USA) was employed for the statistical data evaluation. The results were expressed as mean standard deviation (SD) (n = 3). The p values less than 0.05 or less than 0.01 are considered significant or highly significant, respectively. All graphs were plotted using OriginPro 8.5.1 (OriginLab Corporation, Northampton, MA, USA). 3. RESULTS AND DISCUSSION 3.1. Effect of single factors 3.1.1. The viscozyme – chitinase enzyme ratio Figure 1. Effect of viscozyme-chitinase enzyme ratio on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. 113 Nguyen Huu Nieu, et al. Figure 1 shows the effect of the viscozyme – chitinase enzyme ratio the PS content. As can be seen from the Figure 1, the PS content increased to its highest value of 35.41 mg/g with an increasing ratio of viscozyme – chitinase enzyme until 3:1, after which a slight reduction was observed. According to previous studies, viscozyme has been regarded as having better cell wall hydrolysis ability compared to chitinase due to the fact that viscozyme is a carbohydratehydrolyzing and multi-active enzyme strongly cleaving bonds in the PS matrix [23]. Meanwhile, chitinase is a hydrolytic enzyme used to degrade chitin by breaking down glycosidic bonds [24, 25, 26]. Therefore, the viscozyme – chitinase enzyme ratio of 3:1 was chosen for subsequent experiments. 3.1.2. Total enzyme volume Figure 2 indicates the effect of total enzyme volume on the PS content. While the volume gradually rose from 60 to 220 L, the change in the PS content was recorded and the largest value of 35.41 mg/g could be obtained when the total enzyme volume was at 100 L. Based on the Michaelis – Menten kinetic equation, the relationship between enzyme concentration and the substrates acts as a key factor in determining the reaction rate of enzyme hydrolysis [27, 28]. When applying the sufficient amount of enzymes, the higher extraction yield could be achieved because of the more effective degradation of cell walls leading to an increase in contact between solvents and target compounds. However, the consumption of high concentration of enzyme might also cause decomposition of PS and enzyme waste as enzymes are expensive [29]. Hence, in this study, the total enzyme volume of 100 µL was selected in order to carry out the next experiments. Figure 2. Effect of total enzyme volume on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. 3.1.3. pH values The effect of pH values on the PS content is presented in Figure 3. It is obvious that as the pH values decreased from 4.0 to 5.0, the PS content gradually increased and reached a peak point at 35.41 mg/g with a pH value of 5.5. This could be explained due to the fact that each enzyme has an optimum pH range. The pH values are responsible for structures of enzymes, the interaction between enzymes and substrates, thereby influencing the rate of enzyme reactions 114 Application of ultrasonic-assisted enzymatic extraction for polysaccharides from Vietnamese … [30]. It has been cited that chitinase function within a working pH range from 5 to 5.5 while that of viscozyme was from 4.5 to 5.5 [31, 32]. As a result, the pH value of 5.5 could be considered as suitable for enzyme activities. When pH values reached over 5.5, the content of PS decreased significantly owing to the deactivation of two enzymes resulting in cell wall destruction [33]. Thus, pH value of 5.5 was considered as optimal for the extraction process. Figure 3. Effect of pH values on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. 3.1.4. Extraction temperature Figure 4. Effect of extraction temperature on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. Figure 4 illustrates the effect of extraction temperature on the PS content. With the elevated temperatures, the PS content increased and reached a maximum value of 53.67 mg/g at the extraction temperature of 45 . Temperature is regarded as one of the central keys in the extraction process because of the influence on diffusion and cavitation effect in the ultrasonic bath [34]. The enhancement of the content of PS at high temperature was due to the increase in numbers of air bubbles formed during an ultrasound. The collapse of air bubbles could create 115 Nguyen Huu Nieu, et al. strong diffusion of micro-eddy currents that increased the drawing efficiency of ethanol and enhanced the mass transfer [35]. Additionally, temperature could reduce the viscosity of solvents and increase enzyme activities resulting in an elevation of the PS content. Nonetheless, the too high temperature could cause the inactivation of enzymes, thermal degradation of target compounds, and dissolving impurities leading to a decrease in the PS content [36]. This result is consistent with reported research [37]. Consequently, the extraction temperature of 45 should be the optimal temperature for the UAEE with the highest yield. 3.1.5. Material – to – solvent ratio Figure 5 gives information about the effect of the solvent-to-material ratio on polysaccharide content. As can be seen in Figure 5, the PS content was enhanced from 15.85 to 59.71 mg/g upon increasing material-to-solvent ratio. When the material-to-solvent ratio was greater than 1:25, no further change in the PS content was observed. In general, an increase in the material-to-solvent ratio would improve solvent volume on the inner and outer regions of the plant component, therefore, contact between the substrate and the solvent as well as the transfer kinetics could be accelerated dramatically. As a result, the solubility of targeted components would be elevated [38, 39]. However, very high the material-to-solvent ratio could lead to an over-cell-wall diffusion distance for the solute, as a result, this would negatively influence the dissolution rate and causing a decrease in the PS content [40, 41]. For this reason, 1:25 was set as the optimum material-to-solvent ratio. Figure 5. Effect of material – to – solvent ratio on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. 3.1.6. Ultrasonic power Figure 6 demonstrates the effect of ultrasonic power on the PS content. With an increase in ultrasonic power from 240 to 720 W, the highest PS content was 59.71 mg/g obtained at an ultrasonic power value of 480 W. From the Figure 6, it could be clearly seen that the acceleration of the PS content could be observed with an elevation of ultrasonic power from 120 to 480 W. According to previous studies, it has been postulated that increasing ultrasonic power leads to the disruption of plant cell walls facilitating the penetration of solvents to dissolve 116 Application of ultrasonic-assisted enzymatic extraction for polysaccharides from Vietnamese … effectively target compounds. When ultrasonic power was over 480 W, the content dramatically reduced. The reason for this phenomenon is that higher ultrasonic power could destroy or alter the structure of polysaccharides [42]. So the ultrasound power of 480 W was considered as the optimal ultrasound power for the extraction process. Figure 6. Effect of ultrasound power on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. 3.1.7. Extraction time Figure 7. Effect of extraction time on the PS content. Dissimilar letters in the same graph indicate significantly different at p < 0.05 using one-way ANOVA. The effect of ultrasonic time on the PS content is depicted in Figure 7. It can be seen in Figure 7 that the PS content increased with the increase of extraction time before the value of extraction time of 30 min was reached, at which the PS content achieved its highest point of 117 Nguyen Huu Nieu, et al. 59.71 mg/g, and then it fell rapidly. Obviously, longer extraction time could enhance PS content as adequate extraction time could support mass transfer kinetics and contact between solvents and target compounds. Nevertheless, over the optimal point of extraction time, the PS content significantly diminished. The reason for this is that prolonged extraction time in an ultrasonic bath could disrupt not only cell walls but also polysaccharide structures leading to a reduction in polysaccharide content [21]. Hence, the present work indicated that the extraction time of 30 min gave the highest PS content. 3.2. Antioxidant activity investigation Figure 8 presents the antioxidant activity of ascorbic acid (a) and the extract (b) at different concentrations in DPPH scavenging assay. The IC50 value of ascorbic acid was 3.024 μg/mL, which was lower than that of the extract with value of 1727.15 μg/mL. It can be concluded from Figure 8 that the extract obtained under optimal conditions did not have good antioxidant activity. However, it is reported that the antioxidant activity of PS extracted from G.lucidum is likely to depend on extraction conditions. Previous research showed that the IC50 value of the PS obtained by hot water extraction method was 150 μg/mL, while that of ascorbic acid was under 100 μg/mL [43]. Because of its reducing properties, PS could reduce DPPH radicals with hydroxyl groups and therefore, act as a potent antioxidant agent [44, 45, 46]. Figure 8. Antioxidant activity of ascorbic acid (a) and the extract (b) at different concentrations in DPPH scavenging assay. 4. CONCLUSIONS In this study, PS was extracted from Vietnamese red G. lucidum using UAEE method, followed by the investigation of seven single-factor experiments namely enzyme ratio between viscozyme and chitinase, total enzyme volume, pH value, extraction temperature, material-tosolvent ratio, ultrasonic power, and extraction time. Based on ultraviolet-visible spectroscopy analysis, the highest PS content could be achieved with a value of 59.71 mg/g under extraction conditions including the enzyme ratio between viscozyme and chitinase of 3:1, total enzyme volume of 100 µL, pH value of 5.5, extraction temperature of 45 material-to-solvent ratio of 1:25, ultrasonic power of 480 W, and extraction time of 30 min. For the antioxidant activity evaluation, the obtained extract was then tested by DPPH radical scavenging method, showing 118 Application of ultrasonic-assisted enzymatic extraction for polysaccharides from Vietnamese … that the IC50 value was 1727.15 µg/mL. As a result, the UAEE method could be regarded as an efficient approach for antioxidant crude polysaccharides content extraction from Vietnamese red G. lucidum. Acknowledgments. This research is funded by Vietnam National University Ho Chi Minh City (VNUHCM) under grant number B2019-20-02. The authors acknowledge the support of time and facilities from Ho Chi Minh City University of Technology (HCMUT), VNU-HCM for this study. CRediT authorship contribution statement. NTKN: Conceptualization, Methodology, Investigation, Writing - original draft. TDD: Methodology, Investigation, Writing - original draft. DHL: Writing review & editing. PLTM: Writing - review & editing. NTTL: Methodology, Investigation, Writing original draft. VHT: Methodology, Investigation, Writing - original draft. NDV: Validation, Formal analysis, Data curation, Supervision. NHT: Investigation, Writing - review & editing. HTTT: Writing review & editing. NHBSL: Resources, Visualization. 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